Hepatitis-C virus testing

ABSTRACT

New styles of hepatitis C virus (HCV), referred to as HCV-3 and HCV-4, have been identified and sequenced. Antigenic regions of HCV-2, HCV-3 and HCV-4 polypeptides have been identified. Immunoassays for HCV and antibodies thereto are described, which allow more complete screening of blood samples for HCV, and allow HCV genotyping.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.12/728,030, filed Mar. 19, 2010, which is a divisional of U.S.application Ser. No. 11/652,862, filed Jan. 12, 2007, now U.S. Pat. No.7,709,006, which is a continuation of U.S. application Ser. No.10/396,964, filed Mar. 25, 2003, now U.S. Pat. No. 7,179,470, which is acontinuation of U.S. application Ser. No. 09/039,130, filed Mar. 13,1998, which is a divisional of U.S. application Ser. No. 08/244,116,filed Jul. 15, 1994, now U.S. Pat. No. 5,763,159, which is acontinuation of PCT Application No. GB 92/02143, filed Nov. 20, 1992,and also claims the benefit of Great Britain Application No. 9124696.7,filed Nov. 21, 1991, and Great Britain Application No. 9213362.8, filedJun. 24, 1992, all of which are herein incorporated by reference intheir entirety and for all purposes.

TECHNICAL FIELD

The present invention relates to the discovery of new types of hepatitisC virus, that we have termed type 3 (HCV-3) and type 4 (HCV-4). Inparticular, it relates to the etiologic agent of hepatitis C virus type3 and 4, and to polynucleotides and immunoreactive polypeptides whichare useful in immunoassays for the detection of HCV-3 and HCV-4 inbiological samples; and also to the use of antigenic HCV-3 and HCV-4specific polypeptides in vaccines.

BACKGROUND OF THE INVENTION

Acute viral hepatitis is a disease which may result in chronic liverdamage. It is clinically diagnosed by a well-defined set of patientsymptoms, including jaundice, hepatic tenderness, and an increase in theserum levels of alanine aminotransferase and aspartate aminotransferase.Serologic immunoassays are generally performed to diagnose the specifictype of viral causative agent. Historically, patients presenting withsymptoms of hepatitis and not otherwise infected by hepatitis A,hepatitis B, Epstein-Barr or cytomegalovirus were clinically diagnosedas having non-A, non-B hepatitis (NANBH) by default.

For many years, the agent of non-A, non-B hepatitis remained elusive. Ithas now been established that many cases of NANBH are caused by adistinct virus termed hepatitis C virus (HCV). European PatentApplication EP-A-0318216 discloses CDNA sequences derived from HCV,polynucleotide probes and polypeptides for use in immunoassays. Furtherinformation is provided in European Application EP-A-0388232.

The HCV genome encodes a large polyprotein precursor, which containsstructural and non-structural regions. The single protein is apparentlycleaved into a variety of proteins after production. Most of thestructural and non-structural proteins have now been identified from invitro RNA translation and expression as recombinant proteins. The C andE regions encode for nucleocapsid structural proteins and for envelopestructural proteins, respectively. At least five additional regionsfollow, which encode for non-structural (NS) protein of undefinedfunction. The organization is believed to be as follows (A. Alberti,Journal of Hepatology, 1991; 12; 279 to 282)

5′                                          3′NCR: C : E1 : E2 : NS1 : NS2 : NS3 : NS4 : NS5Certain immunoreactive proteins have been described as recombinantproteins, for example C22 (in the core region), C33 (in NS3 region),5-1-1 and C100 (both in the NS4 region), and NS5 (NS5 region). Diagnosisof hepatitis C is still largely based on methods which detect antibodiesagainst the product of the C-100 clone. This clone was ligated withoverlapping clones to produce a larger viral antigen (C100)corresponding to part of the NS3-NS4 genomic region. C100 was then fusedwith the human superoxide dismutase (SOD) gene, expressed in use as alarge recombinant fusion protein (C100-3) and used on solid phase todevelop radio-labelled (RIA) and enzyme-linked immunosorbent assays(ELISA).

Polynucleotides useful for screening for HCV are disclosed in EuropeanPatent Specification EP-A-0398748. European Patent SpecificationEP-A-0414475 purports to disclose the propagation of HCV in culturecells and the production of antigens for use in diagnostics. EuropeanPatent Specification EP-A-0445423 discloses an improved immunoassay fordetecting HCV antibodies.

Blood banks in the United Kingdom have recently begun routine testing ofblood donors for antibodies to components of HCV. One assay involves thedetection of HCV antibodies to C100-3 polypeptides. The C100-3 antibodyrecognizes a composite polyprotein antigen within non-structural regionsof the virus and is a consistent marker of HCV infection. However, inacute infections this antibody is unreliable because of the delay(typically 22 weeks) in seroconversion after exposure. Furthermore, theC100-3 antibody test lacks specificity for the hepatitis C virus.

Second generation antibody tests employ recombinant antigens orsynthetic linear peptides representing structural antigens from thehighly conserved core region of the virus as well as non-structuralantigens. However, it is found that some second-generation ELISA testscan yield false-positive reactions. The recombinant immunoblot assay(RIBA-2) incorporating four antigens from the HCV genome, provides amethod for identifying genuine-anti-HCV reactivity. However, the resultcan be “indeterminate.” The present workers have reported (The Lancet,338; Oct. 19, 1991) varying reactivity of HCV-positive blood donors to5-1-1, C100, C33C and C22 antigens, and compared these with the resultsof the direct detection of HCV RNA present in the blood samples usingpolymerase chain reaction (PCR) to amplify HCV polynucleotides. However,the work demonstrates that the unambiguous diagnosis of HCV infectionsis not yet possible.

Recently there has been discovered a second type of HCV (References 1,2) called K2 that differs considerably in sequence from the publishedprototype (Reference 3) or the first type K1 sequences (References 4 and5).

SUMMARY OF THE INVENTION

The present invention is based on the discovery of previously unknowntype 3 and 4 variants of HCV, by a comparison to sequences amplified byPCR in certain regions of the HCV genome and confirmed by phylogeneticanalysis. The invention has thus identified polynucleotide sequences andpeptides which are HCV-3 and HCV-4 specific. These may be used todiagnose HCV-3 and HCV-4 infection and should thus be included in anydefinitive test for HCV infection.

One aspect of the invention provides polynucleotide sequences unique tohepatitis C virus types 3 and 4 (HCV-3 and HCV-4). The sequences may beRNA or DNA sequences. In principal any HCV-3 or HCV-4 specificpolynucleotide sequence from non-coding, core, E1, E2 or NS1-5 genomeregions can be used as a hybridization probe. The sequences may berecombinant (i.e. expressed in transformed cells) or synthetic and maybe comprised within longer sequences if necessary. Equally, deletions,insertions or substitutions may also be tolerated if the polynucleotidemay still function as a specific probe. Polynucleotide sequences such ascore, NS3, NS4 and NS5 which code for antigenic peptides areparticularly useful.

Another aspect provides an antigenic HCV-3 or HCV-4 specific peptide,particularly from the core, NS3, NS4 or NS5 regions (e.g. the HCV-3 orHCV-4 counterparts of C100 peptide, 5-1-1 peptide, C33 peptide or C22peptide or epitopes thereof) or peptides including these antigens.

The peptide may be a fusion peptide which comprises at least two of theantigenic HCV-3 or HCV-4 specific peptides. A fusion peptide may alsocomprise at least one of the antigenic peptides fused toβ-galactosidase, GST, trpE, or polyhedron coding sequence.

A further aspect of the invention provides labelled antigenic HCV-3 orHCV-4 specific peptide (or mixtures thereof, particularly from the coreand NS4 regions) for use in an immunoassay.

A further aspect of the invention provides antibodies to HCV-3 or HCV-4specific antigens, particularly monoclonal antibodies for use in therapyand diagnosis. Thus labelled antibodies may be used for in vivodiagnosis. Antibodies carrying cytotoxic agents may be used to attackHCV-3 or HCV-4 infected cells.

A further aspect of the invention provides a vaccine comprisingimmunogenic HCV-3 or HCV-4 specific peptide.

The HCV-3 or HCV-4 specific polynucleotide sequences may be used foridentification of the HCV virus itself (usually amplified by PCR) byhybridization techniques.

Oligonucleotides corresponding to variable regions in the NS-4 regioncould be used for type-specific PCR. Outer sense and inner sense primersmay be used in combination with the two conserved anti-sense primers fora specific detection method for HCV types 1, 2, 3 and 4.

Immunoreactive HCV-3 or HCV-4 specific peptides (particularly from thecore and NS4 regions) may be used to detect HCV-3 and HCV-4 antibodiesin biological samples, and may also provide the basis for immunogens forinclusion in vaccines (especially the E1 polypeptide). The term“peptide” is used herein to include epitopic peptides having the minimumnumber of amino acid residues for antigenicity, oligopeptides,polypeptides and proteins. The peptide may be a recombinant peptideexpressed from a transformed cell, or could be a synthetic peptideproduced by chemical synthesis.

In particular, the invention allows blood donor screening byconventional assays (using HCV type 1 encoded antigens) to besupplemented with a second test that contains two oligopeptidescorresponding to first and second antigenic regions found in the NS-4sequence of HCV type 3 (positions 1691 to 1708; sequenceKPALVPDKEVLYQQYDEM (SEQ ID NO:1) and positions 1710 to 1728; sequenceECSQAAPYIEQAQVIAHQF (SEQ ID NO:2) and two derived from the equivalentregions of HCV type 2, R(A/V)V(V/I)(A/T)PDKE(I/V)LYEAFDEM (SEQ ID NO:3or 4) and ECAS(K/R)AALIEEGQR(M/I)AEML (SEQ ID NO:5 or 6).

The corresponding HCV-4 antigens from substantially positions 1691 to1708 and 1710 to 1728 may be used for HCV-4 detection.

Thus, the present invention has also identified correspondingpolynucleotide and peptide sequences which may be used to identifyhepatitis C type 2 viral infection.

Production and detection of the antigen-antibody immune complex may becarried out by any methods currently known in the art. For example, alabelling system such as enzyme, radioisotope, fluorescent, luminescentor chemiluminescent labels may be employed, usually attached to theantigen. Labelled anti-antibody systems may also be used. Therecombinant antigen may be either used in liquid phase or absorbed ontoa solid substrate.

Oligopeptides corresponding to the antigenic regions of all three majortypes may also be used separately to serologically distinguishindividuals infected with different HCV types. Such an assay could be inthe format of an indirect enzyme immunoassay (EIA) that used sets ofwells or beads coated with peptides of the two major antigenic regionsfor HCV types 4, 3 (SEQ ID NO:1 or 2) and 2 (SEQ ID NO:3, 4, 5 or 6),and with type 1 (KPA(V/I)IPDREVLYREFDEM (SEQ ID NO:7 or 8) andRPAV(I/V)PDREVLYQEFDEM (SEQ ID NO:9) and ECSQHLPYIEG(M/A)AEQF) (SEQ IDNO:10 or 11). Minor degrees of cross-reactivity, should they exist, canbe absorbed out by dilution of the test serum in a diluent thatcontained blocking amounts of soluble heterologous-type oligopeptides,to ensure that only antibody with type-specific antibody reactivitybound to the solid phase.

Immunogens for use in vaccine formulations may be formulated accordingto techniques currently known in the art, including the use of suitableadjuvant and immune-stimulation systems.

Furthermore, the present invention also encompasses assay devices orkits including peptides which contain at least one epitope of HCV-3 orHCV-4 antigen (or antibodies thereto), as well as necessary preparativereagents, washing reagents, detection reagents and signal producingreagents. The antigen may be from the core or NS4 regions. The assaydevice may be in the form of a plate having a series of locationsrespectively containing HCV-1, HCV-2, HCV-3, and optionally HCV-4,specific antigens.

The invention also provides a method of in vitro testing for HCV whichcomprises reverse transcribing any HCV polynucleotide present andamplifying by polymerase chain reaction (PCR), and detecting theamplified HCV polynucleotide employing an HCV-2, HCV-3 or HCV-4 specificpolynucleotide probe.

The invention further provides a method of in vitro HCV typing whichcomprises carrying out endonuclease digestion of an HCV-containingsample employing ScrFI or HaeIII/RsaI endonuclease; and comparing therestriction patterns with characteristic type-specific patterns.

The endonuclease digestion may also employ Hinf1 in a separate or thesame digestion.

The invention furthermore provides a method of in vitro HCV typing whichcomprises carrying out endonuclease digestion of an HCV-containingsample employing ScrFI endonuclease, the restriction pattern beingcharacteristic of HCV-1, HCV-2 and HCV-3; carrying out endonucleasedigestion employing Hinf1 endonuclease, the restriction pattern beingcharacteristic of HCV-4.

DESCRIPTION OF THE DRAWINGS

Examples of the invention will now be described by way of example only.

FIGS. 1A to 1H give cDNA sequences obtained from PCR amplification of aregion −255 to −62 of the 5′ non-coding region of HCV samples from 18blood donors and a comparison with previously published nucleotidesequences (see Table 2); sequence numbering corresponding to theprototype HCV-1 sequence (ref 4) and previous designations of type 1 or2 being indicated: samples E-b1 through E-b8 represent HCV-3 sequences(SEQ ID NO:12).

FIG. 2 is a phylogenetic analysis showing clustering of the sequencesinto three types viz; HCV-1, HCV-2 and HCV-3 for the 5′ NCR results ofFIG. 1 using the maximum likelihood algorithm, shown as an unrootedtree. Numbers 1-18 in full circles correspond to blood donor sequencesE-b1 through E-b18. Numbers 1 to 26 in open circles correspond to thepreviously published sequences identified in Table 2.

FIG. 3 is a comparison of deduced amino acid sequences in the NS-5region of blood donors (E-b1, E-b2, E-b3, E-b7 (type 3) (SEQ ID NO:13)and E-b12 (type 2) with those previously published (Table 2). Amino acidresidue numbering follows that of the HCV-1 polyprotein (4) and usessingle letter amino acid codes.

FIG. 4 is a phylogenetic analysis of the NS-5 region using the maximumlikelihood algorithm, shown as an unrooted tree. Symbols are asdescribed for FIG. 2.

FIG. 5 is a comparison of deduced amino acid sequences in the NS-3region of blood donors (E-b1, E-b2, E-b6, E-b7 (type 3) (SEQ ID NO:14)with those previously published (Table 2). Group 1/1: amino acidsequence of f1, f3, f4, f5, h2, h3, h4 (one), i2, i3, i4, p1, p2; Group1/2: amino acid sequence of i5; Group 1/3: amino acid sequence of h2,h3, h4 (one), h5, f2, p3, i1; Group 1/4: amino acid sequence of h1(one); Group 1/5: amino acid sequence of h1 (one). Numbering, symbolsand abbreviations are as described for FIG. 3.

FIG. 6 is a phylogenetic analysis of the NS-3 region using the maximumlikelihood algorithm, shown as an unrooted tree. Representativenucleotide sequences of the 5 groups of type 1 sequences shown in FIG. 5coded as follows: 19 (full circle) i3; 20 (full circle) i4; 21 (fullcircle) h5; 22 (full circle) h3; 23 (full circle) h1. Symbols are asdescribed for FIG. 2.

FIGS. 7A and 7B are a comparison of deduced amino acid sequences in thecore region of blood donor E-b1 (type 3) (SEQ ID NO:15) with thosepreviously published (Table 2). Numbering, symbols and abbreviations areas described for FIG. 3.

FIG. 8 is a phylogenetic analysis of the core region using the maximumlikelihood algorithm, shown as an unrooted tree. Symbols are asdescribed for FIG. 2.

FIGS. 9A and 9B (SEQ ID NO:16) show nucleotide, and FIG. 9C (SEQ IDNO:17) shows deduced amino acid sequences of HCV type 3 variantsamplified from 5 Scottish blood donors (nos. 40, 38, 36, 26 and 1787) inthe putative NS-4 region of HCV (nucleotides and amino acid residuesnumbered as in Choo et al., (1991). Nucleotide codes: G: guanidine; C:cytidine; A: adenine; U: uridine; amino acid codes: A: alanine; R:arginine; N: asparagine; D: aspartic acid; C: cysteine; Q: glutamine; E:glutamic acid; G: glycine; H: histidine; I: isoleucine; L: leucine; K:lysine; M: methionine; F: phenylalanine: P: proline; S: serine; T:threonine; W: tryptophan; Y: tyrosine; V: valine. “.”: sequence notdetermined; difference from consensus shown in bold.

FIG. 10A shows a comparison of amino acid sequences between residues1679 and 1768 (Choo et al., 1991) of the three major variants of HCV.T16, T42, T77, T1801, T1825: Scottish blood donors infected with HCVtype 1; T351: Scottish blood donor infected with HCV type 2: T59, T940,T810: Scottish blood donors infected with HCV type 2: T40, T38, T36,T26, T1787: Scottish blood donors infected with HCV type 3 (residues 42to 128 of SEQ ID NO:17); and FIG. 10B shows the derivation of consensussequences for HCV types 3 (residues 42 to 128 of SEQ ID NO:17), 2 and 1oligopeptide series. Differences from consensus shown in bold. Aminoacid codes: A: alanine; R: arginine; N: asparagine; D: aspartic acid; C:cysteine; Q: glutamine; E: glutamic acid; G: lycine; H: histidine; I:isoleucine; L: leucine; K: lysine; M: methionine; F: phenylalanine; P:proline; S: serine; T: threonine; W: tryptophan; Y: tyrosine; V: valine;“.”: not determined.

FIGS. 11A to 11F show amino acid sequences of nonameric oligopeptidesused for epitope mapping, derived from consensus HCV type 3 (residues 42to 128 of SEQ ID NO:17), type 2 and type 1 sequences respectively. Aminoacid codes: A: alanine; R: arginine; N: asparagine; D: aspartic acid; C:cysteine; Q: glutamine; E: glutamic acid; G: glycine; H: histidine; I:isoleucine; L: leucine; K: lysine; M: methionine; F: phenylalanine; P:proline; S: serine; T: threonine; W: tryptophan; Y: tyrosine; V: valine;

FIGS. 12A, 12B and 12C show antibody reactivity of three sera from blooddonors infected with HCV type 3 with HCV type 3-encoded oligopeptides inthe antigenic region of NS-4 (sequences 1-82 shown in FIG. 11 a)(derived from residues 42 to 128 of SEQ ID NO:17). Antibody reactivityto oligopeptides (x-axis), recorded as optical densities in the rangefrom −01 to 0.75 (and >0.75) recorded on the y-axis.

FIGS. 13A to 13D are a comparison of divergent HCV sequences withrepresentative type 1, 2 and 3 (SEQ ID NO:18) sequences in variableregions of the 5′NCR. Sequences from −255 to −246, −215 to −186, −115 to−102 and −69 to −62 identical to prototype sequence. “.”: sequenceidentity with HCV-1; “.”: gap introduced in sequences to preservealignment; “−”: sequence not determined. Origins of sequences: Eg-1-33:Egypt; NL-26: Holland; HK-1-4: Hong Kong; IQ-48: Iraq; XX-96: xxxxx.Figures in parentheses number each non-identical sequence.

FIG. 14 is a phylogenetic analysis of the 5′NCR region using the maximumlikelihood algorithm, shown as an unrooted tree. Sequences 1-17 in solidcircles are numbered as in FIG. 13; previously published sequencesnumbered as in table 1 of (992). Scottish blood donor sequencesEb-1-Eb-12 numbered 51-62 in hollow circles. For clarity, onlynon-identical sequences are shown in tree; e.g. Sequence 1 correspondsto those found in samples Eg-16 and Eg-29 etc. (FIG. 1). Hollow squaresare published sequences from Zaire; Hollow small circles are sequencesfrom South Africa; Hollow small solid circles are sequences obtainedelsewhere in the world.

FIGS. 15A and 15B are a comparison of HCV types 1, 2, 3 (SEQ ID NO:19)and 4 (SEQ ID NO:20) nucleotide (A) and HCV types 1, 2, 3 (residues 1 to89 of SEQ ID NO:15) and 4 (SEQ ID NO:21) amino acid (B) sequences in thecore region. Symbols as for FIG. 13. Single letter amino acid codes areused.

FIG. 16 is a phylogenetic analysis of part of the core region using themaximum likelihood algorithm, shown as an unrooted tree. Sequences arenumbered as in FIG. 14; sequence 30 is that of HC-J8 (Okamato et al.Virology 188: 331-341).

FIGS. 17A and 17B show cleavage patterns for A) HaeIII/RsaI and B) ScrFIin 5′NCR.

DETAILED DESCRIPTION

I. Analysis of Hepatitis C Virus and Phylogenetic Relationship of Types1, 2 and 3

Introduction

Sequence analysis of the 5′ non-coding region of hepatitis C virus (HCV)amplified from the plasma of individuals infected in Britain revealedthe existence of three distinct groups of HCV, differing by 9-14% innucleotide sequence. Two of the groups identified were similar to thoseof HCV variants previously termed type 1 and type 2, while the thirdgroup appeared to represent a novel virus type. Sequence comparisonswere then made between the three virus types in other regions of theviral genome. In the NS-5 region, a high degree of nucleotide and aminoacid sequence diversity was observed, with samples classified here astype “3” (SEQ ID NO:13) again forming a distinct group that wasphylogenetically distinct from type 1 and type 2 variants. Type 3sequences were similarly differentiated in the NS-3 (SEQ ID NO:14) andcore (SEQ ID NO:15 and 19) regions from HCV type 1 sequences. Thedesignation of virus types, including an observed sub-division of type 1sequences into geographically distinct variants is discussed in relationto the new sequence data obtained in this study.

Discussion

Replication of nucleotide sequences by polymerase chain reaction (PCR)is a recently established technique. Synthetic complementary primersequences are hybridized to single-stranded DNA on either side of agenome region to be copied. The second strand is built up under theaction of a heat-stable polymerase in the region between the primers.Heating then dissociates the two-strands and the replication processstarts again. The PCR technique allows tiny amounts of polynucleotide tobe amplified provided that there is sufficient sequence information tosynthesize the primer sequences.

The major problem associated with the use of the PCR to assess sequencevariation using the PCR is the possibility that mismatches between theprimers and the variant sequence will prevent amplification. We haveused several strategies to overcome this problem. For initial virusdetection, we used primers in the 5′NCR, which are reported to be highlyconserved amongst type 1 variants (4, 11, 13, 16, 23, 24, 26, 33), andbetween K1 and K2 (23). Sequence analysis of the blood donors allowedthe identification of type 1 and type 2 variants by comparison withpublished sequence data. This analysis also revealed the existence of athird “type” of HCV (SEQ ID NO:12) that appeared to be as distinct fromtype 1 as type 2 was (FIGS. 1, 2; Table 3). Based on our initialtentative classification, we sought corroboration of our findings inother (coding) and more variable regions of the viral genome.

Analysis of the NS-5 region, which was based on several sequences ofeach of the three types (FIGS. 3, 4; Table 3), conformed the existenceof 3 major groups, with type 3 sequences (SEQ ID NO:13) forming arelatively homogeneous group that was quite distinct from types 1 and 2.The proposed separation of type 1 sequences into PT and K1 “sub-types”and type 2 sequences into K2a and K2b is supported by this analysis, inwhich the single type 2 blood donor sequence obtained in this studyappears most similar to K2b. Differential n of HCV type 1 sequences intotwo groups is also clearly shown in the core (FIG. 7) and NS-3 regions(FIG. 5), in both cases with the type 3 sequences (SEQ ID NO:15 and 14,respectively) appearing considerably more distant.

The clustering of phylogenetically distinct groups, their mixeddistributions in a single geographic area (1, 7, 23, 27, 35) and our ownfinding of dual or triple infections in individual hemophiliacs allstrongly suggest that the three types described here are distinctviruses rather than simply representing geographical orepidemiologically clustered variants of a single, highly variable butmonophyletic group.

Our own phylogenetic analysis of the 5′NCR reveals the existence ofthree distinct groups. This contrasts with analyses of coding region,where there appears to be a very prominent differentiation of type 1sequences into two “subtypes”. However, unlike type 2 and 3 variants,the two subtypes are geographically distinct, one sub-type comprisingsequences obtained exclusively from Japanese patients, and the othercomprising predominantly USA/European sequences (Table 2). Indeed theonly exception to this geographical classification is the HC-J1 sequence(26); one apparent exception (Pt-1) was obtained from a Japanesehemophiliac treated with imported factor VIII of USA origin (7, 23),which is likely to have contained HCV variants corresponding to theother sub-type. There is insufficient sequence data to indicate whetherthe two proposed type 2 subtypes, K2a and K2b (7, 23) representgeographically distinct variants.

The genomic organization of HCV corresponds to that of flaviviruses andpestiviruses, with a single open reading frame encoding a polyproteinthat is subsequently cleaved into structural and non-structuralproteins. Weak sequence homologies have been detected with several othervirus groups that have positive-sense RNA genomes (19, 21). Although theoverall degree of sequence dissimilarity between types 1, 2 and 3 cannotbe measured by comparison of the small regions of sequence analyzed inthis study, a rough estimate of the extent of divergence in proteincoding regions is given by an examination of the divergence of thepartial core sequence. This shows that the difference between HCV type 1and type 3 (SEQ ID NO:15) core region (approximately 10% amino acidsequence divergence) is comparable to that which exists betweendifferent serotypes of the flavivirus, tick-borne encephalitis virus(14%; ref. 20), but lower than that which is found between serotypes ofa mosquito borne flavivirus, dengue fever virus (33%), and the West Nile(WN) subgroup (28-43% divergence). The 5′NCR sequences of the differentmembers of WN subgroup are also considerably more diverse than those ofthe three types of HCV (=50% similarity; ref. 5), although within eachof the members e.g. Murray Valley encephalitis virus, the 5′NCR isextremely well conserved (>95% similarity; ref 5). On the basis of theseanalogies, we speculate that the major types of HCV represent distinct“serotypes,” each capable of human infection irrespective of the immuneresponse mounted against other HCV types.

Methods

Samples. Plasma from 18 different blood donors (E-b1 through E-b18),that were repeatedly reactive on screening by Abbott 2nd generationenzyme immunoassay (EIA), and confirmed or indeterminate by arecombinant immunoblot assay (RIBA; Ortho; ref 1) were the principalsamples used in this study. Sequences in the NS-3 region from 5 anti-HCVpositive IVDUs (abbreviated as i1-i5 in ref. 31), 5 hemophiliacs who hadreceived non-heat treated clotting concentrate, and who were alsoanti-HCV positive (h1-h5), 3 pools of 1000 donations collected in 1983(p1-p3), and 5 separate batches of commercially available non-heattreated factor VIII (f1-f5) correspond to those described previously(31).

Primers. The primers used for cDNA synthesis and polymerase chainreaction (PCR) are listed in Table 1 (SEQ ID NO:22 through 43). Theywere synthesized by Oswel DNA Service, Department of Chemistry,University of Edinburgh.

RNA Extraction and PCR. HCV virions in 0.2-1.0 ml volumes of plasma werepelleted from plasma by ultracentrifugation at 100,000 g for 2 hours at4° C. RNA was extracted from the pellet as previously described (2, 31).First strand cDNA was synthesized from 3 μl of RNA sample at 42° C. for30 min. with 7 units of avian myeloblastosis virus reverse transcriptase(Promega) in 20 μl buffer containing 50 mM Tris-HCl (pH 8.0), 5 mMMgCl₂, 5 mM dithiothreitol, 50 mM KCl, 0.05 μg/μl BSA, 15% DMSO, 600 μMeach of dATP, dCTP, dGTP and TTP, 1.5 μM primer and 10 U RNAsin(Promega).

PCR was performed from 1 μl of the cDNA over 25 cycles with eachconsisting of 25 sec. at 94° C., 35 sec. at 50° C. and 2.5 min. at 68°C. The extension time for the last cycle was increased to 9.5 min. Thereactions were carried out with 0.4 unit Taq polymerase (NorthumbriaBiologicals Ltd.) in 20 μl buffer containing 10 mM Tris-HCl, pH 8.8, 50mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 33 uM each of dATP, dCTP, dGTPand dTTP and 0.5 μM of each of the outer nested primers. One μl of thereaction mixture was then transferred to a second tube containing thesame medium but with the inner pair of nested primers, and a further 25heat cycles were carried out with the same program. The PCR productswere electrophoresed in 3% low melting point agarose gel (IBI) and thefragments were detected by ethidium bromide staining and UVillumination. For sequence analysis, single molecules of cDNA wereobtained at a suitable limiting dilution at which a Poisson distributionof positive and negative results was obtained (30).

Direct Sequencing of PCR Products. The PCR products were purified byglass-milk extraction (“GeneClean1”; Bio101, Inc.). one quarter of thepurified products was used in sequencing reactions with T7 DNApolymerase (Sequenase; United States Biologicals) performed according tothe manufacturer's instructions except that the reactions were carriedout in 10% DMOS and the template DNA was heat denatured before primerannealing.

Phylogenetic Methods. The sequences were compiled by version 2.0 of theprograms of Staden (32) and analyzed by programs available in theUniversity of Wisconsin Genetics Computer Group sequence analysispackage, version 7.0 (6). Phylogenetic trees were inferred using twodifferent programs available in the PHYLIP package of Felsenstein(version 3.4 June 1991; ref. 9). The program DNAML finds the tree of thehighest likelihood (the maximum likelihood tree) given a particularstochastic model of molecular evolution and has been shown to performwell in simulation studies (28). In the analyses performed here theglobal (G) option was used as this searches a greater proportion of allpossible trees. The second program used was NEIGHBOR which clusters(following the algorithm of Saitou & Nei: ref 29) a matrix of nucleotidedistances previously estimated using the program DNADIST (which itselfwas set, using the D option, to use the same stochastic model asunderlies DNAML in order to estimate distances corrected for theprobabilities of multiple substitution). In all cases the maximumlikelihood and neighbor joining procedures produced congruent trees andthus only the former have been presented here.

To establish the interrelationships of the major types of HCV, we haveseparately analyzed several regions of the viral genome that differ insequence variability and evolutionary constraint. Thus the conclusionsdrawn from the sequence comparisons are not subject to spuriousevolutionary phenomena that may affect a particular region. However, oneproblem with the analysis presented here was the absence of a viralsequence that was sufficiently distantly related to HCV to serve as anout-group. Thus, although we describe the interrelationships ofdifferent sequence variants of HCV, it should be stressed that we haveno means of deciding which sequence is ancestral to the others. Thetrees are thus drawn in the less familiar un-rooted form to indicatethis.

Results

1) Analysis of the 5′ non-coding region. Samples were obtained from 18blood donors that were repeatedly reactive in the Abbott 2nd Generationenzyme immunoassay and which were confirmed or indeterminate in theChiron 4-RIBA (E-b1 through E-b18, ref 10). HCV sequences present instored plasma samples from each donor were amplified with primerscorresponding to sites in the 5′NCR (SEQ ID NO:22 through 25) (12, 25)that are well conserved between all known HCV type 1 and type 2 variants(4, 11, 13, 16, 23, 24, 26, 33). Sequencing of the PCR product, afterlimiting dilution to isolate single molecules of CDNA beforeamplification, allowed approximately 190 bps in the centre of the regionto be compared with equivalent published sequences (FIG. 1).

Within the sequences, constant as well as variable regions can be found.Six sequences from donors E-b13 through E-b18 closely resembled thosepreviously described as type 1 (4, 11, 13, 16, 23, 24, 26, 33) andothers resembled type 2 (23) sequences (E-b9 through E-b12). However,eight sequences (E-b1 through E-b8) were distinct from both types, andhave been provisionally termed type 3 (SEQ ID NO:12). Division of thesequences into three types is supported by formal phylogenetic analysisusing the maximum likelihood (FIG. 2) and neighbor joining algorithms(data not shown) of the blood donor sequences along with previouslypublished sequences (identified in Table 2). Sequence variability withinthe three groups is in each case considerably less than that whichseparates the types. No sequence intermediate between the three typeswere found. This tree shows that the provisionally identified type 3group (SEQ ID NO:12) is equally distinct from type 1 as is type 2. Usingthe DNAML model, the corrected distances between sequences within eachtype were in each case less than 3%. Between groups, they ranged from 9%(between type 1 and type 3 (SEQ ID NO:12), and between type 1 and 2), to14% between type 2 and type 3 (SEQ ID NO:13) (Table 3).

2) Analysis of the NS-5 Region. The nucleotide sequence of the NS-5region has been found to vary significantly between the previouslydescribed K1 and K2 variants of HCV (7). To investigate whether type 3(SEQ ID NO:13) sequences were equally distant from the other two typesin this region as well as in the 5′NCR, we compared sequences from fourtype 3 blood donors (E-b1, E-b2, E-b3 and E-b7) and one type 2 donor(E-b12) with previously published sequences (FIG. 3; FIG. 4; Table 3).

A remarkable variation was observed between sequences of the three typesin this region. Again, type 3 sequences (SEQ ID NO:13) form a separategroup from type 1 and type 2 in this region. However, unlike the 5′NCR,there appear to be subdivisions within the type 1 and type 2 groups.Type 1 sequences are split between those found in Japanese infectedindividuals (e.g. HCV-J; HCV-BK; sequence numbers 12, 13, 16-20 in Table2) and those of USA origin (HCV-1, Pt-1, H77, H90; sequence numbers 1-4;FIG. 4). There is also some evidence for a split between type 2sequences, those corresponding to their previous designation as K2a (7)appearing distinct from type K2b sequences and the Scottish blood donor,E-b12.

Table 3 shows that the average nucleotide distances between the twogroups of HCV type 1 sequences is 25% (indicated here as type 1a [USA]and type 1b [Japanese]), with variation of only 4-7% within each group.The nucleotide sequence divergence within the two type 1 groups issimilar to that which exists between K2a and K2b (Table 3). However,both of these distances are considerably less than those which existbetween type 1 and type 2 sequences (52-62%), and type 3 (SEQ ID NO:13)(48-49%), and the distance between type 2 and type 3 (SEQ ID NO:13)sequences (53-60%).

3) Analysis of the NS-3 region. Amplification reactions were carried outusing previously published primer sequences in the NS-3 region (37), anda pair of empirically derived inner primers (SEQ ID NO:28 and 29) (31).Although these primers amplified HCV sequences from a high proportion ofanti-C-100 positive sera from hemophiliacs (31), they were lesseffective with sera from IVDUs (31), and with blood donor samples (3positive out of 15 tested; data not shown). Two conserved sites in theamplified fragment were identified by sequence analysis of the NS-3region from the hemophiliac and IVDU patients, and two new primerscorresponding to these were specified (207 (SEQ ID NO:31), 208 (SEQ IDNO:30); Table 1). The combination of 288 (SEQ ID NO:28)-208 (SEQ IDNO:30) (first round) and 290 (SEQ ID NO:29)-207 (SEQ ID NO:31) (secondround) primers successfully amplified samples from four donors infectedwith HCV type 3 (E-b1, E-b2, E-b6 and E-b7) but none of those infectedwith HCV type 2 (data not shown). This enabled a comparison of the newtype (SEQ ID NO:14) with our own (31) and previously published type 1sequences (FIGS. 5, 6; Table 3). For clarity, only seven of the type 1sequences obtained in this study (E-b16, E-b17, i3, i3, h5, h3 and h1)are shown in the tree. These sequences are representative of the rangeof variation found in this region in individuals infected in Britain;comparison of the tree previously published (31) with FIG. 6 shows thatthe former forms a very small component of the overall tree obtainedonce Japanese type 1 and type 3 sequences are added.

The maximum likelihood tree shows that type 1 and type 3 (SEQ ID NO:14)have diverged considerably from each other. As was found in the NS-5region, subtypes of type 1 sequences are found in NS-3. Again, sequencesof Japanese origin (HCV-J, HCV-BK and JH) are distinct from theprototype (PT) sequence, and those found in Scottish blood donors(E-b16, E-b17, p1-3), IVDUs (i1-5) and hemophiliacs (h1-5), all of whichcorrespond to the prototype sequence (FIG. 5). However, the averagesubtype difference (23%) is lower than those that exist between HCV-1and HCV-J with the four type 3 sequences (SEQ ID NO:14) (37-43%). Asreported previously (31), the majority of nucleotide substitutions thatexist between type 1 sequences are silent (i.e. do not affect theencoded amino acid sequence), while numerous amino aced substitutionsexist between type 1 and type 3 (SEQ ID NO:14) sequences (FIG. 5). Theanalysis of the NS-3 region includes the sequence of clone A (35) whichwas obtained from Japanese patients with NANB hepatitis, and which wasreported to be distinct from existing HCV type 1 sequences. In FIG. 6,this sequence appears to be distinct from both HCV type 1 and type 3(SEQ ID NO:14), with corrected sequence distances of 33-43% and 36%respectively. Although it is not possible to assign this sequence to anyknown group at this stage, these distances are not inconsistent with thehypothesis that it represents a type 2 sequence, or an equally distinctnovel HCV type.

4) Partial Sequence of the Putative Core Region of HCV. The regionencoding the putative core protein is comparatively well conserved inits nucleotide sequence between known type 1 variants, showingnucleotide and amino acid sequence similarities of 90-98% and 98-99%respectively (11, 24). Part of the core region from the blood donor Eb1,who has type 3 sequences in other regions analyzed was amplified withprimers 410 (SEQ ID NO:26) and 406 (SEQ ID NO:27) and compared withpreviously published type 1 sequences (FIGS. 7, 8; Table 3). Thisanalysis confirms that the type 3 sequence (SEQ ID NO:15) was distinctfrom those of type 1, and again there was a prominent subdivision oftype 1 sequences into Japanese (HCV-J, HCV-BK, HC-J4, JH and J7) andUSA/European (HCV-1, H77, H90, GM1, GM2) sequences. As was found inNS-3, very little amino acid sequence variation is found in the coreregions of type 1 sequences; almost all of the nucleotide differencesbetween the two groups are at “silent” sites. By contrast, the type 3sequence (SEQ ID NO:15) shows 7-8 amino acid substitutions on comparisonwith type 1 sequences.

TABLE 1SEQUENCES AND SOURCES OF PRIMERS USED FOR AMPLIFICATION OF HCV GENOMEPosition Name Region of 5′base′ Sense^(b) Sequences 5′-3′ Ref. 209 5′NCR8 − ATACTCGAGGTGCACGGTCTACGAGACCT (SEQ ID NO: 22) (12) 211 5′NCR −29 −CACTGCTCGACCCTATCAGGCAGT (SEQ ID NO: 23) (12) 939 5′NCR −297 +CTGTGAGGAACTACTGTCIT (SEQ ID NO: 24) (25) 940 5′NCR −279 +TTCACGCAGAAAGCGTCTAG (SEQ ID NO: 25) (25) 410 CORE 410 −ATGTACCCCATGAGGTCGGC (SEQ ID NO: 26) 406 CORE −21 +AGGTCTCGTAGACCGTGCATCATGAGCAC (SEQ ID NO: 27) 288 NS-3 4951 −CCGGCATGCATGTCATGATGTAT (SEQ ID NO: 28) (31) 290 NS-3 4932 −GTATTTGGTGACTGGGTGCGTC (SEQ ID NO: 29) (31) 208 NS-3 4662 +TCITGAATTTTGGGAGGGCGTCTT (SEQ ID NO: 30) 207 NS-3 4699 +CATATAGATGCCCACITCCTATC (SEQ ID NO: 31) 007 NS-4 5293 −AACTCGAGTATCCCACTGATGAAGTTCCACAT (SEQ ID NO: 32) 220 NS-4 5278 −CACATGTGCITCGCCCAGAA (SEQ ID NO: 33) HCV type 3:¶ 221 NS-4 4858 +GGACCTACGCCCCITCTATA (SEQ ID NO: 34) 008 NS-4 4878 +TCGGTTGGGGCCTGTCCAAAATG (SEQ ID NO: 35) HCV type 2: 281 NS-4 4858GGTCCCACCCCTCTCCTGTA (SEQ ID NO: 36) 509 NS-4 4878CCGCITGGGTTCCGTTACCAACG (SEQ ID NO: 37) HCV type 1: 253 NS-4 4858GGGCCAACACCCCTGCTATA (SEQ ID NO: 38) 196 NS-4 4878CAGACTGGGCGCCGTTCAGAATG (SEQ ID NO: 39) 242 NS-5 8304 −GGCGGAATTCCTGGTCATA000TCCGTGAA (SEQ ID NO: 40) (7) 555 NS-5 8227 −CCACGACTAGATCATCTCCG (SEQ ID NO: 41) 243 NS-5 7904 +TGGGGATCCCGTATGATACCCGCTGCTTTGA (SEQ ID NO: 42) (7) 554 NS-5 7935 +CTCAACCGTCACTGAACAGGACAT (SEQ ID NO: 43) ^(a)Position of 5′base relativeto HCV genomic sequence in ref. no. (4) ^(b)Orientation of primersequence (+: sense: −: anti-sense) ‡Abbreviations: A: adenine. C:cytidine: G: guanidine. T: thymidine. ¶Separate sense primers requiredto enable amplification of each HCV type

TABLE 2 SOURCE AND CITATION OF PREVIOUSLY PUBLISHED HCV SEQUENCES USEDIN THIS STUDY Geographical Ref. No Type Abbreviation Source ReferenceNo. 1 1 HCV-1 U.S.A Choo et al., 1991 (4)  2 1 Pt-1 Japan Nakao et al.,1991 (23) Enomoto et al., 1990 (7)  3, 4 1 H77. H90 U.S.A Ogata et al.,1991 (24) 5.6 1 GM-1. GM-2 Germany Fuchs et al., 1991 (11) 7 1 11 JapanHan et al., 1991 (13) 8 1 A1 Australia Han et al., 1991 (13) 9 1 S 1 S.Africa Han et al., 1991 (13) 10 1 T1 Taiwan Han et al., 1991 (13) 11 1U18/I24 U.S.A/Italy Han et al., 1991 (13) 12 1 HCV-J Japan Kato et al.,1990 (16) 13 1 HCV-BK Japan Takamizawa et al., 1991 (33) 14-15 1 HC-J1.4Japan Okamoto et al., 1990 (26) 16-20 1 Kl. Kl-1-4 Japan Enomoto et al.,1990 (7)  21 1 JH Japan Kubo et al., 1990 (17) 22 1 J7 Japan Takeuchi etal., 1990 (34) 23-26 2 K2a. K2a-1 Japan Nakao et al., 1991 (23) 02 b.K2b-1 Enomoto et al., 1990 (7)  27 ? Clone A Japan Tsukiyama-Kohara 1991(35)

TABLE 3 NUCLEOTIDE DISTANCES BETWEEN THE THREE HCV TYPES IN FOUR REGIONSOF THE GENOME. REGION TYPES (n1) Ia 1b 2a 2b 3 5′NCR 1 (20) 0.0163 n/a^(b) 2 (6) 0.0869 n/a 0.0214 3 (8) 0.0948 n/a 0.1331 n/a 0.0123 CORE1a (6) 0.0358 1b (5) 0.0855 0.0227 3 (1) 0.1801 0.1511 n/d^(c) n/d0.0000 NS-3 1a (34) 0.0699 1b (3) 0.2270 0.0535 3 (4) 0.3689 0.4279 n/dn/d 0.0460 NS-5 1 a (4) 0.0743 1b (7) 0.2477 0.0372 2a (2) 0.6092 0.62060.0612 2b (3) 0.5214 0.5732 0.2252 0.0655 3 (4) 0.4754 0.4890 0.59830.5299 0.0322 ^(a)number of sequences analysed ^(b)n/a: not applicable^(c)n/d: not doneII. Serological Reactivity of Blood Donors Infected with Three DifferentTypes of Hepatitis C Virus

HCV sequences were amplified in the 5′ non-coding region (5′NCR), core,NS-3 and NS-5 regions from blood donors, hemophiliacs and intravenousdrug abusers.

Blood donations that were repeatedly reactive on screening with Abbott2nd generation enzyme immunoassay (EIA) and positive or indeterminate byOrtho recombinant immunoblot assay (RIBA) were amplified by primers inthe 5′NCR (ref 10). The first fourteen PCR-positive blood donations(where PCR was used to amplify and thus detect HCV RNA present in theblood) were then typed by sequence analysis of the amplified region, andcompared with their serological reactivity to a range of structural andnon-structural peptides in two 1st generation EIAs (Ortho HCV ELISA;Abbott HCV EIA) and two RIBA assays (Ortho RIBA and Innogenetics LIA;Table 4). The five donations containing HCV type 1 sequences werepositive in both EIAS, reacted with all antigens in the Ortho RIBAassay, and were broadly reactive in the LIA. However, all but two of thesera from donors with type 2 and 3 infections were completely negativean anti-C100 EIA screening and failed to react with 5-1-1, C100 (RIBA)and NS4 (LIA).

Furthermore, some carriers of HCV type 3 variants reacted poorly withthe C33 (NS-3) peptide in the Ortho RIBA, and yielded two“indeterminate” results (donor nos. 11 and 13).

Thus, current tests using Ortho RIBA and (to a lesser extent)Innogenetics LIA tests are unable to reliably detect HCV-2 and HCV-3genotypes. For reliable testing for all HCV types, antigens from 5-1-1,C100 and NS4 for each of the three types of HCV should preferably beincluded in the panel of antigens.

TABLE 4 SEROLOGICAL REACTIVITY OF SERA FROM BLOOD DONORS INFECTED WITHTHREE TYPES OF HEPATITIS C VIRUS Donor HCV Anti C100 Ortho RIBAInnogenetics LIA Number genotype 0 A† 5-1-1 C100 C33 C22 NS4 NS5 C1 C2C3 C4 E-b13 1 + +  3§ 4 4 4  2§ 3 1 2 1 1 E-b15 + + 4 4 4 4 2 3 3 2 2 1E-b16 + + 4 4 4 4 2 3 2 3 3 − E-b17 + + 4 4 4 4 3 3 3 2 1 1 E-b18 + + 44 4 4 3 − 2 1 1 − E-b9 2 + + − 1 3 4 − − 3 1 1 3 E-b10 − − − − 4 4 − 3 22 2 − E-b11 − − − − 4 4 − 3 4 2 2 3 E-b12 − − − − 4 4 − 1 3 1 2 2 E-b1 3− − − − − 4 − 1 3 1 − 3 E-b2 − − − − 4 4 − 2 1 1 1 2 E-b3 + + − − 2 4 22 1 2 2 1 E-b5 − − − − 2 4 − − 3 1 2 3 E-b7 − − − − − 4 − 2 3 1 1 4*Ortho HCV ELISA (Recombinant C100-3) †Abbott HCV EIA (Hepatitis CRecombinant DNA Antigen) ‡Core oligopeptides. 1-4 §Bands scored −(negative) to 4 (strong-positive) according to manufacturersinstructions.III. Mapping of Antigenic Determinants in NS-4Introduction

With an overall aim of improving serological screening assays, we haveobtained sequence data from the antigenic region of region correspondingto c100-3 for types 2 and 3. This information was used to epitope mapthe region, to define additional immunoreactive peptides that could beused to improve serological anti-HCV assays.

Methods

PCR and sequencing. Plasma samples from Scottish blood donors yieldingrepeatedly reactive donations on 2nd generation anti-HCV screening(Abbott or Ortho), and which were confirmed or indeterminate onconfirmatory testing by RIBA (Chiron) were referred to the Department ofMedical Microbiology from the Scottish National Blood TransfusionService Microbiology Reference Laboratory. HCV RNA within the plasmasamples was extracted and amplified with primers in the 5′NCR asdescribed previously (Chan et al., 1992). HCV was typed by sequenceanalysis of the amplified DNA as described previously (Simmonds et al.,1990) and by RFLP analysis.

Five samples from different donors infected with HCV type 3 (nos. 40,38, 36, 26 and 1787), four infected with type 2 (nos. 31, 59, 940 and810) and five with type 1 infection (nos. 16, 42, 77, 1801 and 1825)were amplified with primers corresponding to sense and anti-sensesequences (SEQ ID NO:32 through 39) spanning the antigenic region ofNS-4 (Table 1). Nucleotide sequences obtained from the amplified DNAwere compared and used to define consensus sequences for each HCV type.In-frame translation of the nucleotide sequences yielded anuninterrupted consensus amino acid sequence that was used to define aseries of overlapping oligopeptides for epitope mapping.

Epitope mapping and determination of antibody specificities. Overlappingsynthetic peptides were synthesized on polypropylene pins using kitscommercially available from Cambridge Research Biochemicals Ltd. Theprinciple of the addition reactions is described in refs (Geysen et al.,1984; Geysen et al., 1985). Antibody reactions were carried out on pinsdisrupted by sonication (30 minutes) in 1% sodium dodecyl sulphate, 0.1%2-mercaptoethanol, 0.1 M sodium dihydrogen orthophosphate. Pins werepre-coated in 1% ovalbumin, 1% bovine serum albumin, 0.1% Tween-20 inphosphate buffered saline (PBS) for one hour at room temperature. Serumor plasma was diluted 1.40 in PBS+0.1% Tween-20 (PBST) and incubatedwith the blocked pins at 4° C. for 18 hours. After washing in 4 changesof PBST (10 minutes at room temperature, with agitation), bound antibodywas detected by incubation in a 1/20000 dilution of affinity isolatedanti-human IgG, peroxidase conjugate (Sigma) for one hour at roomtemperature. Following washing (4 changes in PBST), pins were incubatedin a 0.05% solution of azino-di-3-ethyl-benzthiazodinsulphonate in 0.1 Msodium phosphate/sodium citrate buffer (pH 4.0) containing 0.03%hydrogen peroxide for 20 minutes. Optical densities were read at 410 nm.

Results

HCV RNA in plasma samples from five donors infected with HCV type 3 bysequence analysis of the 5′NCR, and by RFLP were amplified in the NS-4region using primers (SEQ ID NO:32 through 39) listed in Table 1.Because of the high degree of sequence variability in this region, itwas necessary to use separate sense primers (SEQ ID NO:34 through 39)for the amplification of different HCV types. However, the anti-senseprimers (SEQ ID NO:32 and 33) were in a highly conserved region andcould be used for amplification of all three types. Sequence analysiswas carried out as previously described. This gave a continuous sequencefrom position 4911 to 5271 (numbered as in Choo et al., 1991) (HCV-3—SEQID NO:16) (FIG. 9A Little sequence variability (highlighted) wasobserved between the four different donors in this region.

The nucleotide sequences were used to deduce the sequence of the encodedpeptide (FIG. 9B). The putative protein contains mainly hydrophilicresidues but no potential sites for N-linked glycosylation. Amino acidsequence variability with HCV type 3 was confined to only five residues(SEQ ID NO:17) (FIG. 9B. However, this region differed considerably fromthe amino acid sequences of other blood donors infected with HCV types 1and 2 (T16, 42, 77, 1801, 1825, 351, 940 and 810; FIG. 10A). Sequencecomparison between the major HCV types from residues 1679 to 1769reveals three regions of considerable amino acid sequence variability.Most of the observed differences between types involve non-synonymousamino acid substitutions, particularly alternation of acidic and basicresidues in the hydrophilic regions. These changes would be expected toprofoundly alter the overall conformation of the protein, and itsantigenicity.

The consensus amino acid sequences in this region of types 1, 2 and 3(SEQ ID NO:17) (FIG. 10B) were used to define three series of 82nonameric oligopeptides (spanning residues 42 to 128 of (SEQ ID NO:17)overlapping by eight of the nine residues with those before and after inthe series (FIG. 11A-C). These were synthesized on a 12×8 arrays ofpolypropylene pins as described in Methods. Antibody reactivity to theimmobilized antigens on the pins was determined by indirect ELISA usingan overnight incubation with a 1/40 dilution of test serum overnight at4° C., followed by washing, and detection with an anti-humanIgG-peroxidase conjugate and appropriate substrate (see Methods).

Reactivity of an anti-HCV negative, PCR-negative donor, with no knownrisk factors for HCV infection with the three series of peptides wasdetermined. No significant reactivity is shown with any of theHCV-encoded oligopeptides. Reactivity of sera from three donors infectedwith HCV type 3 (derived from residues 42 to 128 of SEQ ID NO:17) toeach of the oligopeptides is shown in FIGS. 12A-12C. All three serareacted with peptides ranging from No. 13 (sequence KPALVPDKE aminoacids 54 to 62 in (SEQ ID NO:17; FIG. 7) to No. 22 (sequence VLYQQYDEM;residues 63 to 71 in SEQ ID NO:17) in the first antigenic region,although the precise peptides recognized varied slightly betweenindividuals. All three sera reacted to varying extents with a secondantigenic region, lying in the range from oligopeptides 32 to 42 (ofsequence ECSQAAPYI, residues 73 to 81 of SEQ ID NO:17, to QAQVIAHQF,residues 83 to 91 of SEQ ID NO:17). Weaker and more variable reactivitywas observed to peptides 48 (residues 88 to 96 of SEQ ID NO:17) to 53(residues 94 to 102 of SEQ ID NO:17). Finally, significant reactivitywas also observed to single oligonucleotides 2 (residues 43 to 51 of SEQID NO:17) (2 of 3 samples), 61 (residues 102-110 of SEQ ID NO:17) (2 of3), 66 (residues 107 to 115 of SEQ ID NO:17) (3 of 3), 73 (residues 114to 122 of SEQ ID NO:17) (3 of 3) and 80 (residues 121 to 129 of SEQ IDNO:17) (2 of 3).

The sequences of the major antigenic regions of HCV type 3 differconsiderably from those encoded by any of the type 1 or type 2 variants.The region bounded by peptides 13 to 22 (SEQ ID NO:1) shows averagehomologies of 50% with HCV type 2 (SEQ ID NO:3 and 4) variants and 67%with type 1 (SEQ ID NO:7 and 8). Between peptides 32 to 42 (SEQ IDNO:2), there are homologies of 39% with type 2 (SEQ ID NO:5 and 6) and58% with type 1 (SEQ ID NO:10 and 11) variants. Thus, although similarregions of each NS-4 sequence are antigenic, the actual epitopes differconsiderably between HCV types.

Discussion

The NS-4 region of HCV type 3 (SEQ ID NO:16 and 17) shows considerablesequence divergence from other variants of HCV, that exceeds that foundin the core, NS-3 or NS-5 regions previously analyzed (Chan et al.,1992). The function of the protein encoded by this region of the HCVgenome is unknown, and the consequences of this variability on virusreplication and pathogenesis are unknown. The function of the NS-4region in flaviviruses and pestiviruses is also poorly defined.

The degree of amino acid sequence variability, and the nature of theamino acid substitutions indicate that the major sites of antibodyreactivity are also those of antigenic variability. This undoubtedlyunderlies the restricted cross-reactivity of HCV type 1 NS-4 encodedantigens with sera from individuals infected with different HCV types.Serological diagnosis of infection is currently based entirely onrecombinant or synthetic oligopeptide sequences derived ultimately fromHCV type 1 sequences (Choo et al., 1991). The serological response toinfection is often very restricted in its initial stages, with antibodyto only one of the recombinant antigens used for screening. Not onlydoes this present difficulties with supplementary antibody tests, wherereactivity to two HCV-encoded antigens is required for confirmation, butcan lead to an increased probability of failing to detect earlyinfection with HCV types 2 and 3.

Table 7 relates HCV typing determined by PCR, using type-specific senseprimers (SEQ ID NO:44 through 49) and the nontype-specific anti-senseprimers (SEQ ID NO:22 and 23) (Table 6), to results obtained usingtype-specific antigens (TSA) and shows good correlation for HCV1-3types.

TABLE 5 SEQUENCES OF NS-4 ENCODED ANTIGENS FOR (A) IMPROVEDSEROLOGICAL DIAGNOSIS. AND (B) FOR SEROLOGICALDISCRIMINATION OF INFECTION WITH DIFFERENT HCV TYPES TypeRegion 1 (1691-1708)* Region 2 (1710-1728) A) 3 KPALVPDKEVLYQQYDEM†(SEQ ID NO: 1) ECSQAAPYIEQAQVIAHQF (SEQ ID NO: 2) 2‡RVVVTPDKEILYEAFDEM (SEQ ID NO: 3) ECASKAALIFEGQRMAEML (SEQ ID NO: 5)RAVIAPDKEVLYEAFDEM (SEQ ID NO: 4) ECASRAALIEEGQRIAEL (SEQ ID NO: 6) B) 3KPALVPDKEVLYQQYDEM (SEQ ID NO: 1) ECSQAAPYIEQAQVIAHQF (SEQ ID NO: 2) 2RVVVTPDKEILYEAFDEM (SEQ ID NO: 3) ECASKAALIEEGQRMAEML (SEQ ID NO: 5)RAVIAPDKEVLYEAFDEM (SEQ ID NO: 4) ECASRAALIIEEGQRIAEML (SEQ ID NO: 6) 1KPAIIPDREVLYREFDEM (SEQ ID NO: 7) ECSQHLPYIEGMLAEQF (SEQ ID NO: 10)KPAVIPDREVLYREFDEM (SEQ ID NO: 8) ECSQHLPYIEGALAEQF (SEQ ID NO: 11)*Amino acrid positions numbered as in Choo et al., (1991). †Amino acidcodes: A: alanine: R: arginine: N: asparagine. D: aspartic acid: C:cysteine: Q: glutamine: E: glutamic acid: G: glycine: H: histidine: I:isoleucine: L: leucine: K: lysine: M: methionine: F: phenylalanine: P:proline: S: serine: T threonine. W: tryptophan. Y: tyrosine: V: valine.‡T Alternative peptides. where there is variability within an HCV type.

TABLE 6 SEQUENCES OF OLIGONUCLEOTIDES SUITABLE FOR DIRECT DETECTION OFHCV TYPE 3 IN CLINICAL SPECIMENS BY POLYMERASE CHAIN REACTION PositionName Region of 5′base* Pol.† Sequences 5′-3′‡ 007 NS-4 5293 −AACTCGAGTATCCCACTGATGAAGTTCCACAT (SEQ ID NO: 32) 220 NS-4 5278 −CACATGTGCTTCGCCCAGAA (SEQ ID NO: 33) Type 3¶ TS-3a NS-4 5140 +GCCGCCCCATATATCGAACA (SEQ ID NO: 44) TS-3b NS-4 5161 +GCTCAGGTAATAGCCCACCA (SEQ ID NO: 45) Type 2: TS-2a NS-4 5140 +AAAGCCGCCCTCATTGAGGA (SEQ ID NO: 46) TS-2b NS-4 5161 +GGGCAGCGGATGGCGGAGAT (SEQ ID NO: 47) Type 1: Type 1: TS-1a NS-4 5140 +CACTTACCGTACATCGAGCA (SEQ ID NO: 48) TS-1b NS-4 5161 +GGGATGATGCTCGCCGAGCA (SEQ ID NO: 49) *Position of 5′ base relative toHCV genomic sequence in Choo et al. (1991). †Orientation of primersequence (+: sense: −: anti-sense) ‡Abbreviations: A: adenine. C:cytidine: G: guanidine. T: thymidine. ¶Type-specific sense primers foramplification of HCV types 3, 2 and 1 variants.

TABLE 7 COMPARISON OF SEROLOGICAL TYPING BY HCV-TSA WITH PCR NumberTYPE-SPECIFIC ANTIBODY PCR^(a) tested 1 2 3 1 + 2 1 + 3 2 + 3 NTS^(b)NR^(c) 1 57 63 — — — 1 — 3 3 2 12 — 11 — — — 1 1 0 3 47  1 — 45 — 2 — 44 Hem^(d) 27 11 —  4 1 4 — 3 4 ^(a)‘Genotype of HCV sequences amplifiedby PCR and typed by RFLP (McOmish et al. 1992) ^(b)NTS: No type-specificantibody detected ^(c)NR: non-reactive with NS-4 peptides ^(d)Samplesfrom HCV-infected hemophiliacs un-typed by PCR.IV. Identification of HCV Type-4Introduction

Investigations were carried out on sequence variations in the 5′non-coding region (5′NCR) of HCV samples from a variety of worldwidegeographical locations (FIG. 13), and also in the core region (FIGS. 15Aand 15B). Phylogenetic analysis (FIGS. 14 and 16) revealed a newdistinct HCV type which we refer to herein as HCV-4.

Methods

Samples. RNA was extracted from plasma samples that were repeatedlyreactive on second generation screening assays for HCV, and which wereeither confirmed (significant reactivity with two or more antigens inthe Chiron recombinant immunoblot assay; Chiron Corporation, Emeryville,Calif., USA) or indeterminate (reactive with only one antigen) fromblood donors and patients with NANBH. Most of the samples containingsequences that differed substantially fcm known HCV types came fromEgypt (EG 1-33). Others came from Holland (NL-26), Hong Kong (HK 1-4),Iraq (IQ-48) and XX (xx-(6).

Sequence determination. HCV sequences were reverse transcribed andamplified with primers matching conserved regions in the 5′NCR aspreviously described (1). For analysis of the core region, RNA wasreverse transcribed using a primer of sequenceCA(T/C)GT(A/G)AGGGTATCGATGAC (SEQ ID NO:50) (5′ base: xxx, numbered asin [20]). cDNA was amplified using this primer and a primer in the 5′NCRof sequence ACTGCCTGATAGGGTGCTTGCGAG (SEQ ID NO:51) (5′ base: −54). Thesecond PCR used primers of sequences AGGTCTCGTAGACCGTGCATCATG (SEQ IDNO:52) (5′ base: −21) and TTGCG(G/T/C)GACCT(A/T)CGCCGGGGGTC (SEQ IDNO:53) (5′ base: xxx). Amplified DNA in both regions was directlysequenced as described previously (ref 1a).

Sequence analysis. Sequences were aligned using the CLUSTAL program inthe University of Wisconsin GCG package (ref. 6). Phylogenetic treeswere constructed by the DNAML program in the PHYLIP package ofFelsenstein (version 3.4, June 1991; (ref 9), using the global option.RNA secondary structures in the 5′NCR of 4 representative HCV variants(refs) were predicted using the program FOLD. Three predictions weremade from each sequence between nucleotides −341 to −1, −341 to +300,and −341 to +900 to allow for possible long range interactions.Comparison of the predicted conformations for each sequence over thedifferent lengths showed that only relatively small scale features, suchas the stem/loop analyzed in the results were at all conserved (data notshown).

All sequences reported in this part have been submitted to GenBank.

Results

Divergent 5′NCR sequences (SEQ ID NO:58). Several sequences in the 5′NCregion detected in samples of blood donors from Saudi Arabia, Hollandand Hong Kong, and from NANBH patients in Iraq and xxx differedsubstantially from those found in Scottish blood donors—and thosereported elsewhere (FIG. 13). Instead of showing the well characterizednucleotide substitutions that distinguish HCV types 1, 2 and 3 from eachother, a new set of sequence differences were observed in the newvariants that appeared to place them outside the existing system ofvirus classification. This can be more simply represented byreconstructing a phylogeny of the sequences and presenting the resultsas an evolutionary tree (FIG. 14). This analysis confirms that sequences1-10 cluster separately from the variants previously typed as 1, 2 and 3(SEQ ID NO:12). For convenience we will refer to sequences within thisnew group as HCV type 4 (SEQ ID NO:18). Mean distances within type 4 andbetween type 4 and the other HCV types in the 5′NCR were comparable tothose previously described for type 1-3. Although sequences within type4 (SEQ ID NO:18) are relatively closely grouped, sequences 11, 12 and 13differ considerably from any of the known types.

Using this phylogenetic tree, it can be seen that the majority ofpreviously published 5′NCR sequences can be readily identified as types1, 2 or 3 (SEQ ID NO:12). Furthermore, almost all of the sequences fromZaire (shown as hollow squares) cluster closely within type 4,suggesting a wider distribution in Africa. However, a furthercomplication is that three identical sequences obtained from SouthAfrican patients appeared distinct from both the type 1 and the type 4(SEQ ID NO:18) group, and may represent yet another HCV type.

RNA from three representative type 4 variants (Eg 29, 33, 21;corresponding to 5′NCR sequences nos. 1-3) was amplified using primersin the core region of HCV polyprotein. All three sequences differedconsiderably at both the nucleotide (SEQ ID NO:20) and amino acid (SEQID NO:21) level from HCV types 1, 2 and 3 (SEQ ID NO:19 and residues 1to 89 of SEQ ID NO:15, respectively) (FIGS. 15A/B). Phylogeneticanalysis of these sequences and those previously analyzed indicated thatthey formed a separate, relatively homogeneous group distinct from theother types (FIG. 16). Reconstructed nucleotide distances between type 4(SEQ ID NO:20) and types 1, 2 and 3 (SEQ ID NO:19) were comparable tothose that exist between the three known HCV types of HCV. Although mostof the nucleotide sequence differences were silent, there were between 4and 9 amino acid differences between the new variants (SEQ ID NO:21) andother types.

V. HCV Typing

Introduction

In view of the sequence variations between HCV types 1, 2, 3 and 4,differences in restriction enzyme cleavage sites exist, leading todifferent endonuclease cleavage patterns. This technique was used toidentify HCV genotypes in blood samples from a variety of sourcesworldwide.

(A) Typing of HCV1-3

Methods

Serum Samples. Samples from blood donors in six countries, Scotland,Finland, Netherlands, Hong Kong and Australia and Japan, were availablefrom routine 2nd Generation anti-HCV ELISA screening (Ortho or Abbott).Donor samples that were repeatedly reactive in the above tests werefurther investigated using a supplementary test (Ortho RIBA: Finland,Netherlands, Australia, Egypt, Abbott Matrix: Hong Kong) or samples weretitered for anti-HCV by ELISA (Japan). Samples that were positive(significant reactivity with two or more HCV antigens (1+ to 4+) orindeterminate (reactivity with one antigen only) in the RIBA test or hada titer of >X 4096 by ELISA (Japan only) were tested for viral RNA byPolymerase Chain Reaction (PCR).

RNA PCR: PCR for the detection of HCV RNA was carried out as previouslydescribed by Chan et al. (ref 1a) using primers in the 5′ non-codingregion (5′NCR) in a nested PCR, with primers 209 (SEQ ID NO:22)/939 (SEQID NO:24) and 211 (SEQ ID NO:23)/940 (SEQ ID NO:25) in first and secondreactions respectively.

HCV TYPING. The existence of relatively conserved patterns ofsubstitutions in the 5′NCR that are characteristic of different HCVtypes provide useful signature sequences for identification of HCVgenotypes. Having compared large numbers of different HCV type 1, 2 and3 sequences, we developed a method that differentiated HCV types 1-3 byrestriction endonuclease cleavage of amplified DNA. However, the 19 type4 sequences would appear as type 1 (electrophoretic types Aa and Ab),and for concurrent studies it has been necessary to modify theconditions to identify the new HCV type. All type 4 sequences showed aT→C change at position −167 (position 78 in SEQ ID NO:18) that creates anovel Hinf1 site that is absent in all type 1 (and type 2) sequences. Incombination with ScrFI, and HaeIII/RsaI, it has now proved possible toidentify the new type reliably in numerous countries in the Middle Eastand elsewhere.

Results

The results are summarized in Table 8 for HCV types 1, 2 and 3. TheEgyptian samples gave aberrant restriction patterns on the single ScrFIdigest and were identified as type 4.

TABLE 8 PREVALENCE OF HCV TYPES IN DIFFERENT COUNTRIES HCV TYPES (%)COUNTRY HCV-1 HCV-2 HCV-3 Scotland 86 (51%) 21 (13%) 60 (36%) Finland 3(25%) 5 (42%) 4 (33%) Netherlands 18 (60%) 7 (23%) 5 (17%) Hong Kong 22(63%) 0 (0%) 0 (0%) Australia 13 (57%) 3 (13%) 7 (30%) Japan 31 (77%) 9(23%) 0 (0%) Egypt 0 (0%) 0 (0%) 0 (0%)(B) Modification of PCR-Based Typing Assay to Detect Infection with HCVType 4 in Clinical SpecimensMethods

Extraction of RNA. RNA was extracted from 100 μl aliquots of plasma ofnon-A, non-B patients by addition of 1 ml RNAzol solution (2Mguanidinium thiocyanate, 12.5 mM sodium citrate [pH 7.0], 0.25% w/vN-lauroylsarcosine, 0.05 M 2-mercaptoethanol, 100 mM sodium acetate [pH4.0], 50% w/v water saturated phenol) as previously described(Chomczynski et al. 1987), and mixed until precipitate dissolved. Afteraddition of 100 μl chloroform, each sample was spun for 5 minutes at14000×g and the aqueous phase re-extracted with 0.5 ml chloroform. RNAwas precipitated by addition of an equal volume of isopropanol andincubation at −20° C. for at least 1 hour. An RNA pellet was produced bycentrifugation at 14000×g for 15 minutes at 4° C., washed in 1 ml 70%cold ethanol solution, dried and resuspended in 20 μldiethylpyrocarbonate treated distilled water. Of the 100 directlyextracted samples, a total of 19 were PCR-negative (see below). Two mlvolumes of the negative samples were ultracentrifuged at 200000×g for 2hours and the pellet re-extracted as described above. Extraction fromthe larger volume of plasma yielded an additional 3 positive samples(numbers 66, 80, 85).

PCR and typing. RNA was reverse transcribed with primer 940 (SEQ IDNO:25) and cDNA amplified in a two stage nested PCR reaction withprimers 940 (SEQ ID NO:25)/939 (SEQ ID NO:24), followed by 209 (SEQ IDNO:22)/211 (SEQ ID NO:23) as previously described (Chan et al. 1992).PCR product was radiolabeled with [³⁵S]-dATP analyzed by restrictionendonuclease cleavage (McOmish et al. Transfusion, 32:no. 11 1992).Samples were cleaved with ScrFI and a combination of HaeIII/RsaI in twoseparate reactions to identify HCV types 1/4, 2, 3. FIG. 17 showsendonuclease cleavage patterns. HCV types 1 and 4 were differentiated bya third reaction with Hinf1 (see Results). Two samples yieldedrestriction patterns that were different from those of the four knowntypes of HCV and were analyzed further by direct sequence analysis ofthe amplified DNA (Chan et al. 1992). These two samples contained 5′NCRsequences distinct from those of known HCV types and currently remainunclassified.

Results

Modification of RFLP method to identify HCV type 4. Previous sequenceanalysis in the 5′NCR of HCV amplified from plasma of Egyptian blooddonors revealed a relatively homogeneous group of novel sequencevariants in both the 5′NCR (SEQ ID NO:18) and core (SEQ ID NO:20 and 21)region which were as distinct from HCV types 1, 2 and 3 (SEQ ID NO:19and residues 1 to 89 of SEQ ID NO:15 as these latter types were fromeach other (see previous submission). This new group was designated asHCV type 4.

Comparison of cleavage patterns of type 4 sequences with those of typeRFLP analysis of the previously identified type 4 sequences produced adistribution of electropherotypes with ScrFI and HaeIII/RsaI similar tothat HCV type 1 (Table 9). Type 1 sequences yielded 9 patterns of aA/B,35 of bA/B and 1 bC. With these enzymes alone, type 4 sequences werethus indistinguishable from type 1 (14 aA/B, 4 bA/B). However, type 1and type 4 sequences consistently differ in the number of Hinf1 sites.All 18 type 4 sequences contain one or two potential cleavage sites(producing patterns band c; Table 5) while none are found in any of the45 type 1 sequences analyzed (pattern a). One of the type 4 sequenceswas further differentiated from type 1 and other HCV types by the lossof a restriction site for RsaI, leading to a new pattern of bandsdesignated h (44, 172, 9, 26; first column, Table 9). Finally, a singlesequence, EG-28 lost two sites to produce bands of 216, 9, and 26 bps(pattern i; Table 9). This sequence was distinct from that of any of theknown HCV types (including type 4) and is shown in the table in thecolumn labelled U (unclassified)

Typing of study subjects. RNA was extracted from 100 samples of patientswith NANB hepatitis and amplified with primers in the 5′NCR. Of these,84 were PCR positive, and enabled HCV typing to be carried out by RFLP.This was initially carried out with HaeIII/RsaI and ScrFl, and allowedthe identification of 10 type 2 and 10 type 3 variants (Table 10).Samples showing electrophoretic patterns aA/B or bA/B were furtheranalyzed by cleavage with Hinf1, yielding 38 samples with pattern a,thus identified as type 1, 22 with pattern b and 2 with pattern c, bothidentified as type 4. Finally, two samples showed the unusual cleavagepatterns h and i with HaeII/RsaI and pattern b with Hinf1, and weretherefore directly sequenced. These two sequences were similar to eachother but were unlike any of the known HCV types, and also distinct fromEG-28, the other sequence showing pattern i with HaeIII/RsaI (Table 10).As they cannot be currently classified, they will be referred to as typeU.

TABLE 9 PREDICTED CLEAVAGE PATTERNS OF PUBLISHED 5′NCR SEQUENCES OF HCVTYPES 1, 2, 3 AND 4 WITH RsaI/HaeIII. ScrFI AND Hinfl Predicted cleavagepattern² HCV type HaeIII/RsaI ScrFI Hinfl 1 2 3 4 U^(b) a A/B  a^(c) 9 —— — — b A/B a 35  — — — — b C a 1 — — — — a A/B  b^(d) — — — 13  — a A/B c^(e) — — — 1 — b A/B b — — — 4 — c D a — 5 — — — d D a — 1 — — — d E a— 2 — — — e D a — 1 — — — e E a — 1 — — — f G b — — 14  — 1 f G c — — 1— g G b — — 8 —  h^(f) A/B b — — — 1  i^(g) A/B b — — — — Cleavagepatterns designated for HaeIII/RsaI and ScrFI as described previously(McOmish et al. 1992). ^(b)Cleavage pattern of an HCV variant ofundesigned type ^(c)Pattern a: uncleaved by Hinfl ^(d)Pattern b: DNAcleaved to generate two fragments of sizes 107 and 142 bps (in order 5′−> 3′) ^(e)Pattern c: DNA cleaved to generate three fragments of 56. 51and 142 bps ^(f)New cleavage pattern for HaeIII/RsaI designated h (bandsof 44 bps. 172 bps. 9 bps. 26 bps) ^(g)New cleavage pattern forHaeIII/RsaI designated i (216 bps. 9 bps. 26 bps)

TABLE 10 IDENTIFICATION OF HCV TYPES 1-4 IN STUDY SUBJECTS BY RFLPANALYSIS OF 5′NCR SEQUENCES WITH RsaI/HaeIII, ScrFI AND Hinfl Observedcleavage pattern Inferred HCV type HaeIII/RsaI ScrFI Hinfl 1 2 3 4 U^(a)a A/B a  2 — — — — b A/B a 36 — — — — a A/B b — — — 16  — a A/B c — — —2 b A/B b — — — 6 — c D n.d. — 7 — — — d E n.d. — 3 — — — f G n.d. — — 7— — g G n.d. — — 3 — — h A/B b — — — — 1 i A/B b — — — — 1 — — — — —TOTALS 38 10  10  24  2 ^(a)Two samples yielded unusual restrictionpatterns with HaeIII/RsaI (h. i). Sequence analysis of the 5′NCR placedthem outside existing HCV classification (samples IQ-48, EG-96).VI. Expression and Assay Etc. Techniques

The present invention also provides expression vectors containing theDNA sequences as herein defined, which vectors being capable, in anappropriate host, of expressing the DNA sequence to produce the peptidesas defined herein.

The expression vector normally contains control elements of DNA thateffect expression of the DNA sequence in an appropriate host. Theseelements may vary according to the host but usually include a promoter,ribosome binding site, translational start and stop sites, and atranscriptional termination site. Examples of such vectors includeplasmids and viruses. Expression vectors of the present inventionencompass both extrachromosomal vectors and vectors that are integratedinto the host cell's chromosome. For use in E. coli, the expressionvector may contain the DNA sequence of the present invention optionallyas a fusion linked to either the 5′- or 3′-end of the DNA sequenceencoding, for example, β-galactosidase or to the 3′-end of the DNAsequence encoding, for example, the trp E gene. For use in the insectbaculovirus (AcNPV) system, the DNA sequence is optionally fused to thepolyhedron coding sequence.

The present invention also provides a host cell transformed withexpression vectors as herein defined.

Examples of host cells of use with the present invention includeprokaryotic and eukaryotic cells, such as bacterial, yeast, mammalianand insect cells. Particular examples of such cells are E. coli, S.cerevisiae, P. pastoris. Chinese hamster ovary and mouse cells, andSpodoptera frugiperda and Tricoplusia ni. The choice of host cell maydepend on a number of factors but, if post-translational modification ofthe HCV viral peptide is important, then an eukaryotic host would bepreferred.

The present invention also provides a process for preparing a peptide asdefined herein which comprises isolating the DNA sequence, as hereindefined, from the HCV genome, or synthesizing DNA sequence encoding thepeptides as defined herein, or generating a DNA sequence encoding thepeptide, inserting the DNA sequence into an expression vector such thatit is capable, in an appropriate host, of being expressed, transforminghost cells with the expression vector, culturing the transformed hostcells, and isolating the peptide.

The DNA sequence encoding the peptide may be synthesized using standardprocedures (Gait, Oligonucleotide Synthesis: A Practical Approach, 1984,Oxford, IRL Press).

The desired DNA sequence obtained as described above may be insertedinto an expression vector using known and standard techniques. Theexpression vector is normally cut using restriction enzymes and the DNAsequence inserted using blunt-end or staggered-end ligation. The cut isusually made at a restriction site in a convenient position in theexpression vector such that, once inserted, the DNA sequences are underthe control of the functional elements of DNA that effect itsexpression.

Transformation of a host cell may be carried out using standardtechniques. Some phenotypic marker is usually employed to distinguishbetween the transformants that have successfully taken up the expressionvector and those that have not. Culturing of the transformed host celland isolation of the peptide as required may also be carried out usingstandard techniques.

The peptides of the present invention may be prepared by syntheticmethods or by recombinant DNA technology. The peptides are preferablysynthesized using automatic synthesizers.

Antibody specific to a peptide of the present invention can be raisedusing the peptide. The antibody may be polyclonal or monoclonal. Theantibody may be used in quality control testing of batches of thepeptides; purification of a peptide or viral lysate; epitope mapping;when labelled, as a conjugate in a competitive type assay, for antibodydetection; and in antigen detection assays.

Polyclonal antibody against a peptide of the present invention may beobtained by injecting a peptide, optionally coupled to a carrier topromote an immune response, into a mammalian host, such as a mouse, rat,sheep or rabbit, and recovering the antibody thus produced. The peptideis generally administered in the form of an injectable formulation inwhich the peptide is admixed with a physiologically acceptable diluent.Adjuvants, such as Freund's complete adjuvant (FCA) or Freund'sincomplete adjuvant (FIA), may be included in the formulation. Theformulation is normally injected into the host over a suitable period oftime, plasma samples being taken at appropriate intervals for assay foranti-HCV viral antibody. When an appropriate level of activity isobtained, the host is bled. Antibody is then extracted and purified fromthe blood plasma using standard procedures, for example, by protein A orion-exchange chromatography.

Monoclonal antibody against a peptide of the present invention may beobtained by fusing cells of an immortalizing cell line with cells whichproduce antibody against the viral or topographically related peptide,and culturing the fused immortalized cell line. Typically, a non-humanmammalian host, such as a mouse or rat, is inoculated with the peptide.After sufficient time has elapsed for the host to mount an antibodyresponse, antibody producing cells, such as the splenocytes, areremoved. Cells of an immortalizing cell line, such as a mouse or ratmyeloma cell line, are fused with the antibody producing cells and theresulting fusions screened to identify a cell line, such as a hybridoma,that secretes the desired monoclonal antibody. The fused cell line maybe cultured and the monoclonal antibody purified from the culture mediain a similar manner to the purification of polyclonal antibody.

Diagnostic assays based upon the present invention may be used todetermine the presence or absence of HCV infection. They may also beused to monitor treatment of such infection, for example in interferontherapy.

In an assay for the diagnosis of viral infection, there are basicallythree distinct approaches that can be adopted involving the detection ofviral nucleic acid, viral antigen or viral antibody. Viral nucleic acidis generally regarded as the best indicator of the presence of the virusitself and would identify materials likely to be infectious. However,the detection of nucleic acid is not usually as straightforward as thedetection of antigens or antibodies since the level of target can bevery low. Viral antigen is used as a marker for the presence of virusand as an indicator of infectivity. Depending upon the virus, the amountof antigen present in a sample can be very low and difficult to detect.Antibody detection is relatively straightforward because, in effect, thehost immune system is amplifying the response to an infection byproducing large amounts of circulating antibody. The nature of theantibody response can often be clinically useful, for example IgM ratherthan IgG class antibodies are indicative of a recent infection, or theresponse to a particular viral antigen may be associated with clearanceof the virus. Thus the exact approach adopted for the diagnosis of aviral infection depends upon the particular circumstances and theinformation sought. In the case of HCV, a diagnostic assay may embodyany one of these three approaches.

In an assay for the diagnosis of HCV involving detection of viralnucleic acid, the method may comprise hybridizing viral RNA present in atest sample, or cDNA synthesized from such viral RNA, with a DNAsequence corresponding to the nucleotide sequences of the presentinvention or encoding a peptide of the invention, and screening theresulting nucleic acid hybrids to identify any HCV viral nucleic acid.The application of this method is usually restricted to a test sample ofan appropriate tissue, such as a liver biopsy, in which the viral RNA islikely to be present at a high level. The DNA sequence corresponding toa nucleotide sequence of the present invention or encoding a peptide ofthe invention may take the form of an oligonucleotide or a cDNA sequenceoptionally contained within a plasmid. Screening of the nucleic acidhybrids is preferably carried out by using a labelled DNA sequence.Preferably the peptide of the present invention is part of anoligonucleotide wherein the label is situated at a sufficient distancefrom the peptide so that binding of the peptide to the viral nucleicacid is not interfered with by virtue of the label being too close tothe binding site. One or more additional rounds of screening of one kindor another may be carried out to characterize further the hybrids andthus identify any HCV viral nucleic acid. The steps of hybridization andscreening are carried out in accordance with procedures known in theart.

The present invention also provides a test kit for the detection of HCVviral nucleic acid, which comprises i) a labelled oligonucleotidecomprising a DNA sequence of the present invention or encoding a peptideof the present invention; and ii) washing solutions, reaction buffersand a substrate, if the label is an enzyme.

Advantageously, the test kit also contains a positive control sample tofacilitate in the identification of viral nucleic acid.

In an assay for the diagnosis of HCV involving detection of viralantigen or antibody, the method may comprise contacting a test samplewith a peptide of the present invention or a polyclonal or monoclonalantibody against the peptide and determining whether there is anyantigen-antibody binding contained within the test sample. For thispurpose, a test kit may be provided comprising a peptide, as definedherein, or a polyclonal or monoclonal antibody thereto and means fordetermining whether there is any binding with antibody or antigenrespectively contained in the test sample. The test sample may be takenfrom any of the appropriate tissues and physiological fluids mentionedabove for the detection of viral nucleic acid. If a physiological fluidis obtained, it may optionally be concentrated for any viral antigen orantibody present.

A variety of assay formats may be employed. The peptide can be used tocapture selectively antibody against HCV from solution, to labelselectively the antibody already captured, or both to capture and labelthe antibody. In addition, the peptide may be used in a variety ofhomogeneous assay formats in which the antibody reactive with thepeptide is detected in solution with no separation of phases.

The types of assay in which the peptide is used to capture antibody fromsolution involve immobilization of the peptide on to a solid surface.This surface should be capable of being washed in some way. Examples ofsuitable surfaces include polymers or various types (molded intomicrotiter wells; beads; dipsticks of various types; aspiration tips;electrodes; and optical devices), particles (for example latex;stabilized red blood cells; bacterial or fungal cells; snores; gold orother metallic or metal-containing sols; and proteinaceous colloids)with the usual size of the particle being from 0.02 to 5 microns,membranes (for example of nitrocellulose; paper; cellulose acetate; andhigh porosity/high surface area membranes of an organic or inorganicmaterial).

The attachment of the peptide to the surface can be by passiveadsorption from a solution of optimum composition which may includesurfactants, solvents, salts and/or chaotropes; or by active chemicalbonding. Active bonding may be through a variety of reactive oractivatible functional groups which may be exposed on the surface (forexample condensing agents; active acid esters, halides and anhydrides;amino, hydroxyl, or carboxyl groups; sulphydryl groups; carbonyl groups;diazo groups; or unsaturated groups). Optionally, the active bonding maybe through a protein (itself attached to the surface passively orthrough active bonding), such as albumin or casein, to which the viralpeptide may be chemically bonded by any of a variety of methods. The useof a protein in this way may confer advantages because of isoelectricpoint, charge, hydrophilicity or other physico-chemical property. Theviral peptide may also be attached to the surface (usually but notnecessarily a membrane) following electrophoretic separation of areaction mixture, such as immunoprecipitation.

After contacting (reacting) the surface bearing the peptide with a testsample, allowing time for reaction, and, where necessary, removing theexcess of the sample by any of a variety of means (such as washing,centrifugation, filtration, magnetism or capillary action), the capturedantibody is detected by any means which will give a detectable signal.For example, this may be achieved by use of labelled molecule orparticle as described above which will react with the captured antibody(for example protein A or protein G and the like; anti-species oranti-immunoglobulin-sub-type: rheumatoid factor: or antibody to thepeptide, used in a competitive or blocking fashion), or any moleculecontaining an epitope contained in the peptide.

The detectable signal may be optical or radioactive or physico-chemicaland may be provided directly by labelling the molecule or particle with,for example, a dye, radiolabel, electroactive species, magneticallyresonant species or fluorophore, or indirectly by labelling the moleculeor particle with an enzyme itself capable of giving rise to a measurablechange of any sort. Alternatively the detectable signal may be obtainedusing, for example, agglutination, or through a diffraction orbirefringent effect if the surface is in the form of particles.

Assays in which a peptide itself is used to label an already capturedantibody require some form of labelling of the peptide which will allowit to be detected. The labelling may be direct by chemically orpassively attaching for example a radiolabel, magnetic resonant species,particle or enzyme label to the peptide; or indirect by attaching anyfor of label to a molecule which will itself react with the peptide. Thechemistry of bonding a label to the peptide can be directly through amoiety already present in the peptide, such as an amino group, orthrough an intermediate moiety, such as a maleimide group. Capture ofthe antibody may be on any of the surfaces already mentioned by anyreagent including passive or activated adsorption which will result inspecific antibody or immune complexes being bound. In particular,capture of the antibody could be by anti-species oranti-immunoglobulin-sub-type, by rheumatoid factor, proteins A, G andthe like, or by any molecule containing an epitope contained in thepeptide.

The labelled peptide may be used in a competitive binding fashion inwhich its binding to any specific molecule on any of the surfacesexemplified above is blocked by antigen in the sample. Alternatively, itmay be used in a non-competitive fashion in which antigen in the sampleis bound specifically or non-specifically to any of the surfaces aboveand is also bound to a specific bi- or poly-valent molecule (e.g. anantibody) with the remaining valencies being used to capture thelabelled peptide.

Often in homogeneous assays the peptide and an antibody are separatelylabelled so that, when the antibody reacts with the recombinant peptidein free solution, the two labels interact to allow, for example,non-radiative transfer of energy captured by one label to the otherlabel with appropriate detection of the excited second label or quenchedfirst label (e.g. by fluorimetry, magnetic resonance or enzymemeasurement). Addition of either viral peptide or antibody in a sampleresults in restriction of the interaction of the labelled pair and thusin a different level of signal in the detector.

A suitable assay format for detecting HCV antibody is the directsandwich enzyme immunoassay (EIA) format. A peptide is coated ontomicrotiter wells. A test sample and a peptide to which an enzyme iscoupled are added simultaneously. Any HCV antibody present in the testsample binds both to the peptide coating the well and to theenzyme-coupled peptide. Typically, the same peptide are used on bothsides of the sandwich. After washing, bound enzyme is detected using aspecific substrate involving a colour change. A test kit for use in suchan EIA comprises: (1) a peptide, as herein defined labelled with anenzyme; (2) a substrate for the enzyme; (3) means providing a surface onwhich a peptide is immobilized; and (4) optionally, washing solutionsand/or buffers.

It is also possible to use IgG/IgM antibody capture ELISA wherein anantihuman antibody is coated onto microtiter wells, a test sample isadded to the well. Any IgG or IgM antibody present in the test samplewill then bind to the anti-human antibody. A peptide of the presentinvention, which has been labelled, is added to the well and the peptidewill bind to any IgG or IgM antibody which has resulted due to infectionby HCV. The IgG or IgM antibody can be visualized by virtue of the labelon the peptide.

It can thus be seen that the peptides of the present invention may beused for the detection of HCV infection in many formats, namely as freepeptides, in assays including classic ELISA, competition ELISA, membranebound EIA and immunoprecipitation. Peptide conjugates may be used inamplified assays and IgG/IgM antibody capture ELISA.

An assay of the present invention may be used, for example, forscreening donated blood or for clinical purposes, for example, in thedetection and monitoring of HCV infections. For screening purposes, thepreferred assay formats are those that can be automated, in particular,the microtiter plate format and the bead format. For clinical purposes,in addition to such formats, those suitable for smaller-scale or forsingle use, for example, latex assays, may also be used. Forconfirmatory assays in screening procedures, antigens may be presentedon a strip suitable for use in Western or other immunoblotting tests.

As indicated above, assays used currently to detect the presence ofanti-HCV antibodies in test samples, particularly in screening donatedblood, utilize antigenic peptides obtained from HIV type 1 only and, asdemonstrated herein, such antigens do not reliably detect other HCVgenotypes. Accordingly, it is clearly desirable to supplement testingfor HIV-1 with testing for all other genotypes, for example, types 2, 3and 4, and also any further genotypes that may be discovered.

To test for a spectrum of genotypes, there may be provided a series ofassay means each comprising one or more antigenic peptides from onegenotype of HCV, for example, a series of wells in a microtiter plate,or an equivalent series using the bead format. Such an assay format maybe used to determine the genotype of HCV present in a sample.Alternatively, or in addition, an assay means may comprise antigenicpeptides from more than one genotype, for example, a microwell or beadmay be coated with peptides from more than one genotype.

It has been found advantageous to use more than one HCV antigen fortesting, in particular, a combination comprising at least one antigenicpeptide derived from the structural region of the genome and at leastone antigenic peptide derived from the non-structural region, especiallya combination of a core antigen and at least one antigen selected fromthe NS3, NS4 and NS5 regions. The wells or beads may be coated with theantigens individually. It has been found advantageous, however, to fusetwo or more antigenic peptides as a single polypeptide, preferably as arecombinant fusion polypeptide. Advantages of such an approach are thatthe individual antigens can be combined in a fixed, predetermined ratio(usually equimolar) and that only a single polypeptide needs to beproduced, purified and characterized. One or more such fusionpolypeptides may be used in an assay, if desired in addition to one ormore unfused peptides. It will be appreciated that there are manypossible combinations of antigens in a fusion polypeptide, for example,a fusion polypeptide may comprise a desired range of antigens from oneserotype only, or may comprise antigens from more than one serotype. Theantigenic peptides from serotypes 2, 3 and 4 are preferably thosedescribed herein.

To obtain a polypeptide comprising multiple peptide antigens, it ispreferred to fuse the individual coding sequences into a single openreading frame. The fusion should, of course, be carried out in such amanner that the antigenic activity of each component peptide is notsignificantly compromised by its position relative to another peptide.Particular regard should of course be had for the nature of thesequences at the actual junction between the peptides. The resultingcoding sequence can be expressed, for example, as described above inrelation to recombinant peptides in general. The methods by which such afusion polypeptide can be obtained are known in the art, and theproduction of a recombinant fusion polypeptide comprising multipleantigens of a strain of HCV type 1 is described in GB-A-2 239 245immunoprecipitation. Peptide conjugates may be used in amplified assaysand IgG/IgM antibody capture ELISA.

The peptide of the present invention may be incorporated into a vaccineformulation for inducing immunity to HCV in man. For this purpose thepeptide may be presented in association with a pharmaceuticallyacceptable carrier.

For use in a vaccine formulation, the peptide may optionally bepresented as part of a hepatitis B core fusion particle, as described inClarke et al. (Nature, 1987, 330, 381-384), or a polylysine basedpolymer, as described in Tam (PNAS, 1988, 85, 5409-5413). Alternatively,the peptide may optionally be attached to a particulate structure, suchas liposomes or ISCOMS.

Pharmaceutically acceptable carriers include liquid media suitable foruse as vehicles to introduce the peptide into a patient. An example ofsuch liquid media is saline solution. The peptide may be dissolved orsuspended as a solid in the carrier.

The vaccine formulation may also contain an adjuvant for stimulating theimmune response and thereby enhancing the effect of the vaccine.Examples of adjuvants include aluminium hydroxide and aluminiumphosphate.

The vaccine formulation may contain a final concentration of peptide inthe range from 0.01 to 5 mg/ml, preferably from 0.03 to 2 mg/ml. Thevaccine formulation may be incorporated into a sterile container, whichis then sealed and stored at a low temperature, for example 4° C., ormay be freeze-dried.

In order to induce immunity in man to HCV, one or more doses of thevaccine formulation may be administered. Each dose may be 0.1 to 2 ml,preferably 0.2 to 1 ml. A method for inducing immunity to HCV in man,comprises the administration of an effective amount of a vaccineformulation, as hereinbefore defined.

The present invention also provides the use of a peptide as hereindefined in the preparation of a vaccine for use in the induction ofimmunity to HCV in man.

Vaccines of the present invention may be administered by any convenientmethod for the administration of vaccines including oral and parenteral(e.g. intravenous, subcutaneous or intramuscular) injection. Thetreatment may consist of a single dose of vaccine or a plurality ofdoses over a period of time.

LITERATURE CITED

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1. A method for detecting HCV infection in a mammal, comprising: a)obtaining a blood sample from a mammal; b) isolating RNA from said bloodsample; c) reverse transcribing said RNA intro cDNA; d) performing PCRon said cDNA using sense and antisense primers specific for HCV, whereinsaid sense and antisense primers are selected from the group consistingof: 1) SEQ ID NO:42 and SEQ ID NO:41; 2) SEQ ID NO:43 and SEQ ID NO:40;and 3) SEQ ID NO:43 and SEQ ID NO:41; and e) determining whether HCVinfection was detected by analyzing the results from said PCR.